Agent for immunochemical tests which contains polymers containing carboxyl groups

ABSTRACT

Agents for immunochemical tests which contain polymers containing carboxyl groups and processes for carrying out such tests are described. These polymers are capable of improving the results obtained with such tests, in that they suppress non-specific reactions and increase the sensitivity of the tests.

This application is a continuation, of application Ser. No. 07/443,312,filed Nov. 30, 1989, abandoned.

The invention relates to an agent for the immunochemical detection andfor the determination of an analysis substance in a biological material,this agent containing a water-soluble polymer containing carboxylgroups.

Known immunochemical test systems use additives of proteins,polysaccharides and/or surfactants which do not participate in theimmunochemical reaction but are suitable for favorably influencing theresult of such a reaction.

For immunoassays, for example ELISA, the incubation media required(buffer solutions, incubation medium, conjugate buffer) must be of acomposition such that non-specific binding of concomitant substances ofthe sample and/or conjugate to the solid phase is prevented. The knownadditives, such as proteins, for example albumin, IgG, casein,hydrolyzed gelatin and derivatives thereof, and mixtures of proteins orhuman or animal sera as well as surfactants, are therefore used in theincubation media.

An incubation medium for solid phase immunochemical tests which containslactoferrin, fetal calf serum, polyoxyethylene 20-sorbitan monolaurate(^(R) Tween 20) and buffer salts is described in German Patent3,638,767.

Additives comprising surfactants from the poloxamer group, for example^(R) Pluronic F 68, and from the poloxamine group, for example ^(R)Tetronic 707 and 1107 , are also described in European Patent A-215,457, the three compounds mentioned proving to be advantageous in comparisonwither ween ^(R) Tween 20.

However, incubation media provided with these additives cannot preventincorrect measurement values which occur, for example, in one-stepsandwich tests which use large sample volumes at high conjugateconcentrations but low conjugate volumes in order to achieve optimumsensitivity.

There was therefore the object of discovering an agent with whichincorrect measurement values can be prevented by incubation with thesample.

It has been found, surprisingly, that water-soluble polymers containingcarboxyl groups are even more suitable than additives of the prior artfor preventing incorrect measurement values and favorably influencing animmunochemical reaction, which has the effect of a higher sensitivity ofthe detection and determination of an analysis substance contained in abiological material, which is also to be referred to as the sample.

These polymers, as a constituent of such an agent, can already bepresent during preparation of the sample for the test.

The invention thus relates to an agent for the detection ordetermination of an analysis substance in a biological materialcontaining immunochemical reactants, at least one of which reacts withthe analysis substance, which moreover contains a water-soluble polymercontaining carboxyl groups.

Suitable water-soluble polymers containing carboxyl groups can beprepared from water-insoluble synthetic polymers which carry ester oranhydride groups, by alkaline hydrolysis. Such water-insoluble polymersare known. Examples of these are polymers which are prepared using, asmonomers, esters of acrylic acid and alkylacrylic acid or maleicanhydride, by themselves or as a mixture with monomers containing nocarboxyl groups (for example methyl vinyl ether, styrene, ethylene,propylene or octadecene). These polymers, referred to as homopolymers oras heteropolymers, are in general obtained by free radicalpolymerization, that is to say by addition of free radical initiators,for example by means of coumaryl hydroperoxide or by means of dibenzoylperoxide, the chain length of the polymer being controlled by the amountof peroxide added, or by the temperature or the addition of a telogen,for example carbon tetrachloride or an agent which collects freeradicals, for example N-acetylcysteine or another thiol.

Reaction products of molecular weight greater than 2000 are in generalreferred to as polymers, and those products having a molecular weight ofless than 2000 are referred to as oligomers or telomers.

Water-soluble synthetic heteropolymers containing carboxyl groups aresuccessfully prepared only if the ratio of monomer carrying carboxylgroups and monomers which contain no carboxyl groups is chosen so thatthe resulting polymer, oligomer or telomer is water-soluble followingalkaline hydrolysis. This ratio can be determined empirically for eachheteropolymer. For example, whereas a heteropolymer prepared from themonomers styrene and maleic anhydride at a molar ratio of 2 to 2.5:1 isstill water-soluble after alkaline hydrolysis, at a ratio above 3:1 itremains water-insoluble after alkaline hydrolysis. Water-solublepolypeptides, containing carboxyl groups, which contain aspartic acid,glutamic acid and if appropriate also neutral aminoacids, which arecommercially available or can be prepared by known methods of peptidesynthesis, are also suitable.

The polysaccharides which are obtainable from natural products andcontain sugar acids, for example pectic acid and galacturonic acid, aremoreover suitable.

The following polymers are preferred:

Polyacrylic acid, polymaleic acid, polymers or telomers of maleicanhydride and methyl vinyl ether or ethylene or propylene or octadecene,the anhydride rings of which are opened by hydrolysis, polyasparticacid, polyglutamic acid and polygalacturonic acid.

Telomers of maleic anhydride and methyl vinyl ether or propylene whichare converted into the water-soluble carboxyl-containing polymers bytreatment with sodium hydroxide solution are particularly preferred.

A large number of agents containing immunochemical reactants and/orbinding partners of bioaffinity are known.

The agents are in general named according to the immunochemicalprocesses for which they are used.

Agents in the sense of the invention are those with which precipitatesas a dispersion or in a gel or agglutinates of particles are produced,or absence thereof is effected, or those in which a color signal orradiation is produced or prevented by the immunochemical reaction.

Agents from the group mentioned last which are preferred are those withwhich solid phase immunochemical tests which are called ELISA (enzymelinked immunosorbent assay) or scintillation assay if a coloration orradiation is produced from an enzyme substrate, solid phaseradioimmunoassay if radiation is produced by a radioactively labeledisotope, or solid phase fluorescence assay if fluorescence is producedby a fluorogen.

Diagnostic agents contain antigens, antibodies or both at the same timeas reactants and other binding partners for the reactants or ofbioaffinity to the analysis substance, for example lectins, complement,protein A or G and derivatized biotin and avidin, it being possible forat least one of the reactants to be labeled, and if appropriate reagentsfor detection of the labeling. One of the reactants can moreover bepresent as a solid phase.

A solid phase in the sense of the invention is a water-insoluble carrierto which one or more reactants are bonded.

Examples of carriers are latex particles, granular, swellable ornon-swellable material, beads, internal surfaces of tubes, microtestplates as a particular embodiment of an arrangement of tubes and alsoporous materials to be described as an absorbent matrix.

One constituent of the agent according to the invention can be a devicefor accommodating a sample, for example a vessel for accommodating asample or the uptake zone, for example an absorbent matrix, for thesample on a so-called "dry chemical" test system. The constituent canalso be an aqueous solution which also contains buffer salts and ifappropriate stabilizing additives, such as proteins or polysaccharides,as substances which likewise stabilize the analysis substance, thepolymer being present in a concentration of 0.01 to 50 g/l, preferably0.1 to 20 g/1 and particularly preferably 0.2 to 2 g/l.

The polymer can also be contained in a device as a constituent of thediagnostic agent in which the immunochemical reaction takes place.

The biological materials also called the sample which contain theanalysis substances are, for example, tissue from biopsis or autopsies,blood, blood cells, serum or plasma, secretions, fluid from an inflamedor non-in-flamed tissue, the disintegration products of tissue andmetabolic excretions.

The invention moreover relates to a process for the immunochemicaldetermination of an analysis substance contained in a biologicalmaterial, which comprises bringing the analysis substance together withthe polymer, if appropriate incubating the mixture in an aqueoussolution and performing an immunochemical determination with the mixtureobtained in this way.

The invention furthermore relates to the use of such a polymer inimmunochemical tests.

The biological materials are brought together with the polymer and canbe stored in this way for a prolonged period of time without theanalysis substance contained therein changing in its immunochemicalproperties.

Immunochemical processes in which one of the reactants is present in asolid phase, the treated material described above being brought togetherwith the solid phase, if appropriate together with other immunochemicalreactants, apart from those of the solid phase and reagents fordetection of the analysis substance, the solid phase then beingseparated from the liquid phase and the analysis substance beingdetermined either in the solid or in the liquid phase, are preferred.

EXAMPLES

1. Preparation of water-soluble carboxyl-containing polymers

1.1 Carboxylate of maleic anhydride-propylene telomer (MPT carboxylate)

5 g of MPT 155, a commercial product from Stickstoffwerke Linz, weresuspended in 500 ml of deionized water. 13 ml of 5 normal sodiumhydroxide solution were then added dropwise in the course of one hour,while stirring. The mixture was stirred for a further 3 hours, a pH of 8being maintained by further addition of 1 normal sodium hydroxidesolution. The solution obtained in this way was left to stand at 20°-25°C. for a further 15 hours before it was used further.

1.2 Carboxylate of maleic anhydride-methyl vinyl ether telomer (MMVTcarboxylate) 5 g of a maleic anhydride-methyl vinyl ether telomerobtainable under the name Gantrez AN179 from Serva, Heidelberg, weretreated as described in 1.1.

2. Preparation of constituents of test kits for determination ofhepatitis B surface antigen (HBs) by the solid phase 2-sideimmunochemical process (sandwich ELISA)

2.1 Microtest plates coated with antibodies against HBs

Microtest plates, that is to say immunoplates II 96 F with round bases(Nunc, Roskilde, Denmark, Article No. 262162) were coated withmonoclonal anti-HBs and immunoglobulin G (IgG) from mice. For this, theIgG was diluted to 2 mg/l in 100 mmol/l sodium bicarbonate of pH 9.6.100 μl of the dilution was introduced into each depression (well) of themicrotest plates. The test plates filled in this way were left at 20° C.for 18 hours and were then washed 3-4 times with 200 μl of a solution of1 g/l of ^(R) Tween 20 in phosphate-buffered physiological salinesolution, pH 7.4, by filling and suction, and the test plates were thendried over silica gel at 20° C.

2.2 Anti-HBs IgG peroxidase conjugate

Monoclonal mouse IgG, directed towards HBs, was reacted withN-gamma-maleimidobutyryl oxysuccinimide (GMBS) as described by Tanamoriet al., 1983 in J. Immunol. Meth. 62, 123-131. 2-Iminothiolanehydrochloride (Sigma, Catalog No. I 6256) was reacted with horseradishperoxidase (POD), obtained from Boehringer Mannheim, Catalog No. 413470,as described by King et al. 1978 in Biochem. 17, 1499-1506. An IgG-PODconjugate was prepared from the GMBs-IgG conjugate and theimmunothiolane-POD conjugate as described by Tanamori. The resultingsolution of the IgG-POD conjugate had a protein content of 1.2 mg/ml.The ratio of POD to IgG was determined as 2.5. The solution was thendiluted to 24 and 6 μg/ml of IgG-POD with a solution which contained 200ml/l of bovine serum defibrinized and delipidized by heating, 1 g/l of^(R) Polygeline, 50 mmol/l of tris, 150 mmol/l of trisodium citrate and0.5 mol/l of NaCl, brought to pH 7.4 with HCl and the resultingsolutions were called 24 μl/ml and 6 μg/ml of anti-HBs-POD.

2.3 TMB substrate preparation

To detect anti-HBs-POD, a substrate system or a substrate preparationcontaining hydrogen peroxide and tetramethylbenzidine (TMB) preparedfrom two stock solutions was used.

Stock solution 1: TMB dihydrochloride was dissolved in doubly distilledwater in a concentration of 5 g/l, that is to say 16 mmol/l whilestirring, and the solution was brought to pH 1.5 with 5 normalhydrochloric acid. Penicillin G was added to this solution in a finalconcentration of 200 mg/l that is to say 0.56 mmol/l while stirring.

Stock solution 2:1.4 ml of glacial acetic acid, 1.5 ml of 1 normal NaOHand 250 mg, that is to say 3 mmol, of H₂ O₂ as a urea-hydrogen peroxideadduct were added to 900 ml of doubly distilled water. After thecomponents had dissolved completely, the solution was made up to 1 withdoubly distilled water.

TMB substrate preparation: One part by volume of stock solution 1 and 10parts by volume of stock solution 2 were mixed with one another.

3. Comparative determinations and results

3.1 Determination of HBs by one-step sandwich ELISA

Some sera which had been determined as HBs-negative by the process ofthe prior art, that is to say the two-step sandwich ELISA, but were notto be evaluated unambiguously in the one-step sandwich ELISA withoutusing a polymer containing carboxyl groups because they producedextinctions greater than 0.04 were included in the determination. Sinceall the samples of a reference panel classified as HBs-negative producedextinctions of less than 0.040 in the two-step sandwich ELISA, thisELISA was evaluated as reliable in respect of avoiding falsely positiveresults. A negative and a positive control were also run in thedetermination. 2 depressions of a microtest plate which had been coatedwith antibodies against HBs as described under 2.1 were used for eachcontrol and for each sample. 25 μl of a solution of 24 μg/ml ofanti-HBsPOD and 25 μl of this solution with the addition of 1 g/l ofMMVT carboxylate were used in groups of 2 depressions. Either 100 μl ofnegative or 100 μl of positive control or 100 μl of sample wereintroduced per depression of each group.

The microtest plate treated in this manner was left to stand at 37° C.for 1 hour. The contents of the depressions were then removed by suctionand the depressions were washed 4 times with in each case 200 μl of asolution of 1 g/l of ^(R) Tween 20 in phosphate-buffered physiologicalsaline solution, pH 7.4, by filling and suction. 100 μl TMB substratepreparation were introduced into each depression and left to stand at20-22° C. for 30 minutes, and 100 μl of 1 normal sulfuric acid were thenadded. The extinctions of the solutions in the depressions were measuredat 450 mm against a blank value of 200 μl of phosphate-buffered salinesolution in a further depression.

3.2 Determination of HBs by the two-step sandwich ELISA, a process ofthe prior art

100 μg of negative control, 100 μl g of positive control and 100 g ofthe samples which were also tested in the one-step sandwich wereintroduced into depressions of a microtest plate coated with antibodiesagainst HBs in accordance with Example 2.1. The plate was left to standat 37° C. for 1 hour. The contents of the depressions were then washed 4times, as described in Example 3.1. 100 μl of a solution of 6 μg/ml ofanti-HBs-POD were then added and the plate was left to stand again at37° C for 1 hour. The contents of the depressions were removed and thedepressions were washed, as described above. The addition of TMBsubstrate preparation and of sulfuric acid and the measurement of theextinction of the solutions in the depressions were carried out in thesame manner as described in Example 3.1.

3.3 Measurement results of the determinations of HBs in accordance withExamples 3.1 and 3.2.

The extinctions obtained from the negative and the positive control andfrom 10 serum samples by the one-step and two-step sandwich ELISA areshown in the table. Values for the upper limit, called the "cut-off" ofthe extinction of samples to be classified as HBs-negative are alsogiven. The "cut-off" was specified as the extinction of the negativecontrol plus an extinction of 0.025.

It can be seen from the table that the one-step sandwich assay giveshigher extinctions for the positive control than the two-step sandwichELISA, but that the samples determined as negative in the two-step assayappear as positive in the one-step assay if the solution which containsthe HBs-POD conjugate contains no addition of MMVT carboxylate.

The one-step sandwich ELISA described containing MMVT carboxylate isreliable, at a high sensitivity, in respect of the decision of whether asample is to be classified as HBs-negative, because the extinctionsmeasured are all below 0.040, the "cut-off", and are not about at 0.040.

In further experiments, it was found that one-step sandwich ELISAs fordetection of HBSs which contain MPT carboxylate, polyacrylic acid,polyaspartic acid and/or polygalacturonic acid give equivalent results.

                  TABLE                                                           ______________________________________                                        Sandwich - ELISA                                                              ______________________________________                                                One-step with                                                                 MMVT carboxylate                                                                           One-step  Two-step                                       ______________________________________                                        Extinctions at 450 nm                                                         negative  0.015          0.014     0.019                                      control                                                                       cut-off   0.040          0.039     0.044                                      positive  1.692          1.514     0.918                                      control                                                                       HBs-negative                                                                  sera                                                                           1        0.018          0.281     0.038                                       2        0.011          0.076     0.018                                       3        0.008          0.072     0.028                                       4        0.005          0.024     0.023                                       5        0.007          0.128     0.028                                       6        0.020          0.017     0.025                                       7        0.030          0.011     0.017                                       8        0.036          0.049     0.015                                       9        0.010          0.056     0.029                                      10        0.036          0.015     0.026                                      ______________________________________                                    

We claim:
 1. An agent for the immunochemical detection or determinationof an analysis substance in a sample of biological materialcomprising:(a) at least one immunochemical reactant being capable ofreacting with the analysis substance and being bonded to a solid phase;and (b) an aqueous solution of a water-soluble polymer containingcarboxyl groups selected from the group consisting of polyacrylic acid,polymaleic acid, polymers or telomers of maleic anhydride and methylvinyl ether or ethylene or propylene or octadecene, the anhydride ringsof which are opened by hydrolysis, and polygalacturonic acid, saidpolymer being present in said aqueous solution in a concentration of 0.1to 50 g/L.
 2. An agent as claimed in claim 1, wherein the polymercontaining carboxyl groups is polyacrylic acid.
 3. An agent as claimedin claim 1, wherein the polymer containing carboxyl groups is obtainedby alkaline hydrolysis of maleic anhydride-propylene polymer.
 4. Anagent as claimed in claim 1, wherein the polymer containing carboxylgroups is obtained by alkaline hydrolysis of maleic anhydride-methylvinyl ether polymer.
 5. An agent for the immunochemical detection ordetermination of an analysis substance in a sample of biologicalmaterial comprising:(a) dry chemical test device coated with awater-soluble polymer containing carboxyl groups selected from the groupconsisting of polyacrylic acid, polymaleic acid, polymers or telomers ofmaleic anhydride and methyl vinyl either or ethylene or propylene oroctadecene, the anhydride rings of which are opened by hydrolysis, andpolygalacturonic acid, said device having been coated with said polymerin a concentration of 0.1 to 50 g/L; and, (b) at least oneimmunochemical reactant being capable of reacting with the analysissubstance.
 6. A process for the immunochemical determination of ananalysis substance contained in a sample of biological material, whichcomprises the steps of:(a) applying an aqueous solution containing theanalysis substance and a water-soluble polymer containing carboxylgroups selected from the group consisting of polyacrylic acid,polymaleic acid, polymers or telomers of maleic anhydride and methylvinyl ether or ethylene or propylene or octadecene, the anhydride ringsof which are opened by hydrolysis, and polygalacturonic acid, saidpolymer being present in a concentration of 0.01 to 50 g/L, to animmunochemical reactant present on a solid phase and being capable ofreacting with the analysis substance; (b) separating the solid phasefrom the liquid phase; and (c) determining the analysis substance eitherin the solid phase or in the liquid phase by using an immunochemicalreactant carrying a detectable label.
 7. A process for theimmunochemical determination of an analysis substance contained in asample of biological material, which comprises the steps of:(a) applyingan aqueous solution containing the analysis substance and awater-soluble polymer containing carboxyl groups, present in a dry formbefore addition to the aqueous solution, selected from the groupconsisting of polyacrylic acid, polymaleic acid, polymers or telomers ofmaleic anhydride and methyl vinyl either or ethylene or propylene oroctadecene, the anhydride rings of which are opened by hydrolysis, andpolygalacturonic acid, in a concentration of 0.01 to 50 g/L in theaqueous solution, to an immunochemical reactant capable of reacting withthe analysis substance and present on a solid phase; and, b) determiningthe amount of analysis substance bound to the immunochemical reactant.8. A process for the immunochemical determination of an analysissubstance contained in a sample of biological material, which comprisesthe steps of:(a) adding to an aqueous solution containing the analysissubstance a dry chemical test device coated with a water-soluble polymercontaining carboxyl groups selected from the group consisting ofpolyacrylic acid, polymaleic acid, polymers or telomers of maleicanhydride and methyl vinyl ether or ethylene or propylene or octadecene,the anhydride rings of which are opened by hydrolysis, andpolygalacturonic acid, said device having been coated with said polymerin a concentration of 0.01 to 50 g/L; and an immunochemical reactantpresent on a solid phase and being capable of reacting with the analysissubstance; (b) separating the solid phase from the liquid phase; and (c)determining the analysis substance either in the solid phase or in theliquid phase by using an immunochemical reactant carrying a detectablelabel.